In addition to their medical importance as pathogens, vaccine vectors and oncolytic agents, poxviruses offer tractable model systems for discovering fundamental molecular and evolutionary mechanisms underlying the arms races between viruses and the host cells they parasitize. These conflicts drive rapid evolution of host and viral proteins and can create species barriers to viral transmission. One such case is the dsRNA-activated protein kinase R (PKR) pathway, which has broad antiviral activity but can be blocked, in a species-specific manner, by a variety of viral factors. Experimental evolution coupled with deep sequencing technology offers a powerful strategy for uncovering new factors and mechanisms underlying the arms race between PKR and its antagonists. For example, serial passage of a vaccinia virus (VV) mutant that encodes an antagonist that only weakly blocks PKR in one cell type yielded progeny viruses that were able to replicate much more efficiently not only in the cells used for the serial passage but also in cells from other species. Further studies revealed that amplification of the gene encoding the weak antagonist or mutations in either of two other VV genes, A24R and A35R, neither of which has previously been implicated as a modulator of the PKR pathway, dramatically improves VV replication in cells from multiple species. The proposed studies aim to elucidate the mechanisms by which mutations in A24R and A35R enable VV overcome the PKR and related host defense systems. Thus, this project will reveal new insights into the functions of these conserved poxvirus genes and into the roles and regulation of the PKR pathway and its VV antagonists. More generally, the results will aid in understanding how large DNA viruses can evolve to overcome host defenses and will have implications for understanding the risks and barriers to cross species transmission of viruses.